Xanthomonas is a genus of Proteobacteria, many of which cause plant diseases that leads to major economic loss. This application describes novel methods using native or genetic engineered bacteriophages to inhibit Xanthomonas infection in the field.
Citrus canker is caused by a bacterial pathogen, Xanthomonas citri subsp. citri (formly X. axonopodis pv. citri, or X. campestris pv. citri), and is one of the most devastating diseases on citrus plants. Infection causes lesions on the leaves, stems, and fruit of citrus trees, including lime, oranges, and grapefruit. Canker significantly affects the vitality of citrus trees, causing leaves and fruit to drop prematurely. A fruit infected with canker is generally too unsightly to be sold.
Citrus canker disease was first recorded in South East Asia in 1827, and it is extremely persistent when it becomes established in an area. Citrus groves have been destroyed in attempts to eradicate the disease. Some areas of the world have eradicated citrus canker and others have ongoing eradication programs, but the disease remains endemic in most areas where it has appeared. Because of its rapid spread, high potential for damage, and impact on export sales and domestic trade, citrus canker is a significant threat to all citrus-growing regions. Many countries like the United States and Brazil are currently suffering from canker outbreaks. The first introduction of citrus canker in Florida was in 1910 on trifoliate rootstock seedlings imported from Japan. The disease spread around the Gulf Coast from Texas to Florida and further north to South Carolina. Citrus canker has been a serious problem in Florida since the last outbreak which began in 1995. This disease is now also present in Japan, South and Central Africa, the Middle East, Bangladesh, the Pacific Islands, some countries in South America. In Florida alone, costs of running eradication program from 1995 through 2005 plus compensation to commercial growers and homeowners for residential citrus destroyed is approaching $1 billion dollars. Under the current citrus canker quarantine instituted by USDA/APHIS at the end of 2013, the interstate movement of citrus plants and plant parts other than fruit remains prohibited in the US. (Gottwald, 2000).
Copper-based products have been to be an effective means of controlling citrus canker. However, copper has been shown to stimulate the growth of mite populations in citrus tree (Mao et. al. 2011). Additionally, copper buildup on the citrus groves and copper-resistant pathogens are also potential problems. Developing a new science-based approach for managing and eradicating citrus canker in the field is emergent and critical.
Bacteriophage (or called “phage” in this application interchangeably) is a naturally occurring virus that that infects and replicates within bacteria. The replication of a bacteriophage may have a “lytic cycle” or a “lysogenic cycle”, and a few viruses are capable of carrying out both. With lytic phages, bacterial cells are broken open (lysed) and destroyed after immediate replication of the phage. As soon as the cell is destroyed, the phage progeny can find new hosts to infect. Lytic phages have been used for over 90 years as an alternative to antibiotics in the former Soviet Union and Central Europe, as well as in France. They are seen as a possible therapy against multi-drug-resistant strains of many bacteria. In contrast, the lysogenic cycle does not result in immediate lysing of the host cell. Lysogenic viral genome will integrate with host DNA and replicate along with it fairly harmlessly, or may even become established as a plasmid. The virus remains dormant until host conditions deteriorate, then the endogenous phages (known as “prophages”) become active. At this point they initiate the reproductive cycle, resulting in lysis of the host cell. As the lysogenic cycle allows the host cell to continue to survive and reproduce, the virus is reproduced in all of the cell's offspring.
A novel filamentous bacteriophage Cf was first isolated from citrus canker pathogen, Xanthomonas citri subsp. citri, by Dai et al. in Taiwan (Dai, et al., 1980) and has been studied for decades. Cf bacteriophage and its variants have several unique characteristics with its host bacteria, making it become a novel reagent to prevent and inhibit citrus canker disease in this application:
(1) Unlike other filamentous single-stranded DNA bacteriophages that replicate independently and separately from the host genomic DNAs, two Cf phage variants (Cf1t and Cf16) can integrate their DNAs into its Xanthomonas host genome (Kuo et al., 1987a, Dai, et al, 1988). Only one copy of Cf1t or Cf16 DNA was stably integrated per host chromosome and become lysogenic, which they replicate together with host genome during bacteria growth (Dai et al., 1987; Kuo et al., 1987b).
(2) In contrast to Cf16, one Cf variant called Cf16-v1 was exceedingly unstable during the integration in host DNA genome, and it produces clear plaques and kills host cells. Only 4% of the infected bacterial cells, rather than 95% as in the case of Cf16, retained the phage genome (Dai, et al, 1987).
(3) The integration region of Cf16-v1 phage and host attachment sites (attP and attB) shared an identical 15-bp “core,” 5′-TATACATTATGCGAA-3′. The sequence characteristics indicate that insertion of Cf16-v1 into host genome was accomplished by a site-specific recombination mechanism (Dai et al., 1988).
(4) A virulent Cf variant called Cf1c, was derived from of Cf1t and has been sequenced. The phage yield is higher than Cf1t, and the infected host bacteria growth is drastically reduced (Kuo et al., 1991). Sequence data revealed mutations located in the upstream region of an open reading frame (ORF165) which might encode a 18.2-kDa protein. When the ORF165 in Cf1t was disrupted, this recombinant phage can kill bacteria and form clear plaques (Shieh, et al., 1991).
(5) After Xanthomonas host genome was integrated with Cf1t phage DNA, the bacteria is “immuned” and is no longer susceptible to another Cf phage infection. A variant, Cf1tv (or Cf-tv1), that only lyses cell and its DNA never integrates into host DNA genome, was also isolated. Cf1tv has been proven able to superinfect the immuned bacteria that already contain Cf inside. After infected with Cf1tv, bacterial cell division was seriously affected and finally stopped (Kuo et al., 1994). The region that causes this superinfection activity of Cf1tv and similar variant Cf-tv2 has also been mapped (Wang et al., 1999; Cheng et al., 1999).
A Cf phage variant named XacF1 that shares 99% sequence homology with Cf1c was also isolated from Xanthomonas citri and reported in Japan in July 2014 (Ahmad, et al., 2014). Infection by XacF1 phage caused several physiological changes to the bacterial host cells, including lower levels of extracellular polysaccharide production, reduced motility, slower growth rate, and dramatic reduction in virulence (Ahmad, et al., 2014) (FIG. 17).
Despite that bacteriophages have been used as a tool to kill bacteria in several studies and patents (U.S. 2010/0068185 A1, 3/2010; U.S. Patent No. 2007/0292395 A1, 12/2007), the specific tool toward to citrus canker disease is unavailable to date. The novel properties make Cf phage and its variants described above become powerful reagents to eradicate citrus canker pathogen in the field.
During our work surveying new phages that can potentially inhibit citrus canker pathogen, a new prophage Cf2 is also identified that integrates within genomic sequence of Xanthomonas citri (strain Aw 12879) and is described in this invention. Cf2 phage DNA integrates within genomic sequence of citrus canker pathogen Xanthomonas citri (strain Aw 12879) and exists as a prophage. The genome of Cf2 prophage is 6453 nucleotide in size, smaller than Cf1c (7303 nucleotide) or Cf variant XacF1 (7325 nucleotide). Cf2 shares 88% nucleotide sequence homology and has similar gene organization with other filamentous phage phiLf, indicating that Cf2 is also a lysogenic ssDNA phage that belongs to Inoviridae family (FIG. 13A).
Sequence analysis reveals that Cf2 phage contains 12 putative protein-coding genes, including Cf1cp7-like protein, chloride channel EriC, Zot protein gI, minor coat protein gVI, gIX and A, major coat protein B, ssDNA binding protein gV, RstA-type replication protein, 2 hypothetical proteins, and a protein similar to Cf 18.2 kDa (ORF165) protein. Importantly, Cf2 shares only very limited sequence homology (less than 10%) with Cf or its variant XacF1 phage, indicating that it is a new lysogenic phage that infects citrus canker pathogen X. citri. 
Unlike Cf or phiLf phage, Cf2 phage contains RstA-type replication initiation protein (FIG. 13A). RstA-type replication initiation protein is also found in other lysogenic phages such as phiSMA6, phiSMA7, and phiSMA9 phages that infect bacterial host Stenotrophomonas maltophilia and Xylella fastidiosa related to Xanthomonas. It suggests that Cf2 may require different host factors for its replication. The amino acid sequence of major coat protein B of Cf2 phage shares 76% identity to phiLf phage (FIG. 13B). Characterization of minor coat protein A of Cf2 exhibits novel chimeric properties. Its C-terminus amino acid 303 to 382 is 73% identical to phiLf coat protein A. However, N-terminal amino acid 3 to 211 of Cf2 coat protein A shares 28% identity to Cf homologue (FIG. 12B). Since host specificity of Cf or phiLf phage for X. citri and X. campestris is mediated by minor coat protein A (Yang and Yang, 1997), this result also suggests that the determinant domain of coat protein A for phage-host specificity is located at its N-terminus.
Cf2 phage DNA also integrates into host bacterial genome at attP sequence located at the C-terminus of 18.2 kDa protein similar to Cf ORF165. Similar location of attP sequence is also found in other lysogenic ssDNA filamentous phages, such as Cf1c, XacF1, phiLf, and Xf (FIG. 14). However, Cf2 contains a different 13-bp core attP sequence (5′-TAATTATGTCAAA-3′) (SEQ ID NO: 64) in comparison to 15-bp core attP sequence (5′-TATACATTATGCGAA-3′) (SEQ ID NO: 63) identified in other lysogenic filamentous phages (FIG. 14).
The region of DNA sequence and organization that causes superinfection activity of Cf1tv and Cf-tv2 is located at the upstream sequence of ORF165 that contains a predicted promoter region encoding cM1 and cM2 transcripts (FIG. 3, FIG. 5B, FIG. 15) (Cheng et al., 1999). The 49 base pair deletion of Cf-tv2 is upstream of ORF 165. The T in the start codon of ORF165 is mutated of the single base substitution to A in Cf1tv. This T is also located next to −10 consensus TATA box of a predicted promoter for cM1 transcript (FIG. 5B). In addition, the virulent Cf variant XacF1 also contains G to A substitution at this −10 TATA box, which is one nucleotide downstream to Cf1tv mutant (Ahmad, et al., 2014) (FIG. 15). These results lead to the conclusion that this predicted promoter is the determinant for phage immunity.
Cf2 and Cf phage shares almost no sequence homology in their entire genome except the starting 58 nucleotides of cM2 transcript of Cf is 95% identical to Cf2. The −35 and −10 consensus sequences are also found in the upstream of ORF162 in Cf2 (FIG. 15), indicating that the critical region for phage immunity is also conserved in Cf2. These findings suggest that Cf2 can be also engineered into a virulent variant as a bio-control reagent against citrus canker pathogen.
This invention describes the applications of Cf and Cf2 and their variant bacteriophages to infect and kill Xanthomonas citri subsp. citri, including producing Cf and Cf2 phages in an industrial laboratory and used in citrus groves.
The infectivity of Cf phages requires its minor coat protein A (Yang and Yang, 1997). When Cf genome loses its coat protein A gene, it no longer produces infectious phage particles, and becomes harmless to its bacterial host. Loss of infectivity in the coat protein A-mutated Cf phage can be completely rescued in the presence of minor coat protein A when co-expressed by the other vector. This provides a great system to generate recombinant Cf phages that cannot infect other new bacteria after killing the first bacterial cell they encounter. Unlike other bacteriophage patents in effect so far, this invention also includes a new method to make recombinant Cf phages as “controllable” reagents and do not spread out in natural environment after treating citrus canker in citrus groves. This method will generate bacteriophages as much more secure anti-bacterial reagents without being harmful to the rest of environment.
In taxonomy, Cf phage belongs to Inoviridae family, in which a group of filamentous phages (e.g., Xf, fd, If1, Ike, Pf1, Pf3, phiLF, etc.) has been characterized for their biochemical and biophysical properties (reviewed in Day et al., 1988). For examples, Xf phage was isolated from rice bacterial blight disease pathogen Xanthomonas oryzae or phiLF from Xanthomonas campestris (Kuo et al., 1969; Tseng, et al., 1990; Weng and Tseng, 1994). Compared to Cf, both Xf and phiLF phage particles are relatively stable. The Xf phage particles are resistant to treatment with nucleases or proteases and also maintain its full infectivity in phosphate buffer (pH 7.0) when stored in −15° C. for one year (Kuo et al., 1969). phiLF phage particles are stable for 6 months at 4° C. and keep 100% infectivity even at 80° C. for more than 10 minutes (Tseng et al., 1990). These stable properties of the coat proteins also has made Xf phage an excellent model virus in many biophysical studies previously (Lin et al, 1971; Martin et al., 1974; Wiseman and Day, 1977; Chen, et al., 1980; Thomas and Day, 1981; Casadevall and Day, 1982; Thomas et al., 1983; Marzec and Day, 1983; Marzec and Day, 1988; Thomas et al., 1988; Marzec and Day, 1994). We take advantage of the fact that Cf and Xf phages can be packed with the coat protein from each other (Yang and Yang, 1997). This invention also includes a method engineering a recombinant Cf phage with the coat proteins from other members of Inoviridae family (Xf, phiLF, fd, If1, Ike, Pf1, Pf3, Cf2 etc.) to enhance the stability of Cf phage particles for further application.
Xanthomonas oryzae pv. oryzae causes rice bacterial blight (BB) disease which is one of the most important diseases of rice in most of the rice growing countries (Nino-Liu, et al., 2006). Rice blight has high epidemic potential and is destructive to high-yielding cultivars in both temperate and tropical regions especially in Asia. Its occurrence in the 70 s in Africa and the Americas has led to concerns about its transmission and dissemination. X. oryzae pv. oryzae can destroy up to 80 percent of a crop if the disease develops early. Even if it develops late, it can nonetheless severely diminish the quality and yield of the grain. Bacterial leaf blight is a prevalent and destructive disease that affects millions of hectares throughout Asia. In Japan alone, annual losses are estimated to be between 22,000 and 110,000 tons. In the Philippines, susceptible varieties lose up to 22.5% of the total harvest during wet seasons and up to 7.2% in the dry season. In resistant crops, these numbers are, respectively, 9.5% and 1.8% (Exconde, 1973).
Research on bacterial blight of rice was commenced in Japan as early as in 1901, and the efforts were focused mainly on ecological studies and chemical control. Since then, significant gains have been made in understanding BB through analysis of the interactions between X. oryzae pv. oryzae and rice at many levels, including studies focused on the epidemiology, population biology, physiology, cell biology, biochemistry, and molecular genetics of the host pathogen interaction. Bacterium oozes from leaf lesions and is spread by wind or rain, especially when strong storms occur and cause wounds to plants. X. oryzae has a wide host range that includes a rice cutgrass called Leersia sayanuka which acts as alternative host for the bacterium. The presence of L. sayanuka, is also key to the spread of disease because it is a naturally growing weed usually found around patties and has the ability to be infected by the bacterium and spread the bacterium through a rice patty.
One virulent bacteriophages Xp12, was isolated from Xanthomonas oryzae pv. oryzae in the irrigation water in a rice field in Taiwan in 1968 (Kuo et al., 1968). Xp12 phage is distinguished from other known phages isolated so far because it processes DNA in which all the cytosine residues are completely replaced by 5-methylcytosine (Kuo et al. 1968; Ehrlich et al. 1975). This 5-methylcytosine substitution has made Xp12 an important tool for studying the mechanism of naturally occurring DNA methylation in molecular biology field for decades (Kuo and Tu 1976; Ehrlich et al. 1977; Wang and Ehrlich, 1982; Kuo et al., 1982). Xp12 has been also a model system for analyzing the digestion ability of restriction endonucleases (McClelland and Nelson 1991).
The other bacteriophage, Xf, is a filamentous phage also able to infect rice blight but not inhibit bacterial growth (Kuo, et al., 1969). Xf phage invades the host cells with its coat protein (Kuo and Lin, 1976). However, the sequence information of Xf phage genome has not been reported. We also identify the complete nucleotide sequence of Xf phage genome. In an aspect, the disclosure applies the unique features of Xp12 and Xf phage to engineer these phages and to the control the rice blight disease in the field.
Xanthomonas campestris pv. citri was reclassified as X. axonopodis in 1995. In 2006, the species designations for pv. citri and malvacearum were revised to X. citri and these pathovars are now referred to as subspecies Xanthomonas campestris.